title: Selective modulation of coupled ryanodine receptors during microdomain activation of calcium/calmodulin-dependent kinase II in the dyadic cleft.
abstract: BACKGROUND
  In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs.
  
  
  OBJECTIVE
  Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation.
  
  
  RESULTS
  We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost.
  
  
  CONCLUSIONS
  Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.
authors.affiliation: From the Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium (E.D., V.B., I.L., G.A., K.R.S., N.M.); Biomedical Research Institute, University of Hasselt, Belgium (V.B.); Division of Cardiology, Medical University of Graz, Austria (G.A.); and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (N.M.).
authors.forename: Eef
authors.initials: E
authors.lastname: Dries
authors.forename: Virginie
authors.initials: V
authors.lastname: Bito
authors.forename: Ilse
authors.initials: I
authors.lastname: Lenaerts
authors.forename: Gudrun
authors.initials: G
authors.lastname: Antoons
authors.forename: Karin R
authors.initials: KR
authors.lastname: Sipido
authors.forename: Niall
authors.initials: N
authors.lastname: Macquaide
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000502:physiology*; D004195:Disease Models, Animal; D021621:Imaging, Three-Dimensional; D021962:Membrane Microdomains / Q000502:physiology*; D018613:Microscopy, Confocal; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D032383:Myocytes, Cardiac / Q000473:pathology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D017382:Reactive Oxygen Species / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D012508:Sarcolemma / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine
chemical_list: D017382:Reactive Oxygen Species; D019837:Ryanodine Receptor Calcium Release Channel; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: NADPH oxidase; calcium/calmodulin-dependent protein kinase type 2; myocytes, cardiac; reactive oxygen species; ryanodine receptor calcium release channel; sarcoplasmic reticulum